Hepatic cell junctions: Pulling a double-duty.

Cell junctions, including anchoring, occluding and communicating junctions, play an indispensable role in the structural and functional organization of multicellular tissues, including in liver. Specifically, hepatic cell junctions mediate intercellular adhesion and communication between liver cells. The establishment of the hepatic cell junction network is a prerequisite for normal liver functioning. Hepatic cell junctions indeed support liver-specific features and control essential aspects of the hepatic life cycle. This review paper summarizes the role of cell junctions and their components in relation to liver physiology, thereby also discussing their involvement in hepatic dysfunctionality, including liver disease and toxicity.

The Helicobacter pylori infection alters the intercellular junctions on the pancreas of gerbils (Meriones unguiculatus).

Helicobacter pylori is a common resident in the stomach of at least half of the world's population and recent evidence suggest its emergence in other organs such as the pancreas. In this organ, the presence of H. pylori DNA has been reported in cats, although the functional implications remain unknown. In this work, we determined distinct features related to the H. pylori manifestation in pancreas in a rodent model, in order to analyse its functional and structural effect. Gerbils inoculated with H. pylori exhibited the presence of this bacterium, as revealed by the expression of some virulence factors, as CagA and OMPs in stomach and pancreas, and confirmed by urease activity, bacterial culture, PCR and immunofluorescence assays. Non-apparent morphological changes were observed in pancreatic tissue of infected animals; however, delocalization of intercellular junction proteins (claudin-1, claudin-4, occludin, ZO-1, E-cadherin, ß-catenin, desmoglein-2 and desmoplakin I/II) and rearrangement of the actin-cytoskeleton were exhibited. This structural damage was consistent with alterations in the distribution of insulin and glucagon, and a systemic inflammation, event demonstrated by elevated IL-8 levels. Overall, these findings indicate that H. pylori can reach the pancreas, possibly affecting its function and contributing to the development of pancreatic diseases.

Comparative Transcriptome Analysis Identifies Desmoglein-3 as a Potential Oncogene in Oral Cancer Cells.

The role of desmoglein-3 (DSG3) in oncogenesis is unclear. This study aimed to uncover molecular mechanisms through comparative transcriptome analysis in oral cancer cells, defining potential key genes and associated biological processes related to DSG3 expression. Four mRNA libraries of oral squamous carcinoma H413 cell lines were sequenced, and 599 candidate genes exhibited differential expression between DSG3-overexpressing and matched control lines, with 12 genes highly significantly differentially expressed, including 9 upregulated and 3 downregulated. Genes with known implications in cancer, such as MMP-13, KRT84, OLFM4, GJA1, AMOT and ADAMTS1, were strongly linked to DSG3 overexpression. Gene ontology analysis indicated that the DSG3-associated candidate gene products participate in crucial cellular processes such as junction assembly, focal adhesion, extracellular matrix formation, intermediate filament organisation and keratinocyte differentiation. Validation of RNA-Seq was performed through RT-qPCR, Western blotting and immunofluorescence analyses. Furthermore, using transmission electron microscopy, we meticulously examined desmosome morphology and revealed a slightly immature desmosome structure in DSG3-overexpressing cells compared to controls. No changes in desmosome frequency and diameter were observed between the two conditions. This study underscores intricate and multifaceted alterations associated with DSG3 in oral squamous carcinoma cells, implying a potential oncogenic role of this gene in biological processes that enable cell communication, motility and survival.

Surface layer proteins from Lactobacillus helveticus ATCC® 15009™ affect the gut barrier morphology and function.

Paraprobiotics and postbiotics represent a valid alternative to probiotic strains for ameliorating and preserving a healthy intestinal epithelial barrier (IEB). The present study investigated the effects of surface layer proteins (S-layer) of the dairy strain Lactobacillus helveticus ATCC® 15009™ (Lb ATCC® 15009™), as paraprobiotic, on the morpho-functional modulation of IEB in comparison to live or heat-inactivated Lb ATCC® 15009™ in an in vitro co-culture of Caco-2/HT-29 70/30 cells. Live or heat-inactivated Lb ATCC® 15009™ negatively affected transepithelial electrical resistance (TEER) and paracellular permeability, and impaired the distribution of Claudin-1, a tight junction (TJ) transmembrane protein, as detected by immunofluorescence (IF). Conversely, the addition of the S-layer improved TEER and decreased permeability in physiological conditions in co-cultures with basal TEER lower than 50 ohmcm2, indicative of a more permeable physiological IEB known as leaky gut. Transmission electron microscopy (TEM) and IF analyses suggested that the S-layer induces a structural TJ rearrangement and desmosomes' formation. S-layer also restored TEER and permeability in the presence of LPS, but not of a mixture of pro-inflammatory cytokines (TNF-α plus IFN-γ). IF analyses showed an increase in Claudin-1 staining when LPS and S-layer were co-administered with respect to LPS alone; in addition, the S-layer counteracted the reduction of alkaline phosphatase detoxification activity and the enhancement of pro-inflammatory interleukin-8 release both induced by LPS. Altogether, these data corroborate a paraprobiotic role of S-layer from Lb ATCC® 15009™ as a possible candidate for therapeutic and prophylactic uses in conditions related to gastrointestinal health and correlated with extra-intestinal disorders.

Role of desmosomal components in the initiation and metastasis of oral cancer-A review.

Desmosomes are composed of a number of proteins, including cadherins, armadillo proteins and plakoplilins, which are responsible for mediating cell-cell adhesion. Cadherins are transmembrane proteins that bind to each other on adjacent cells, forming a strong adhesive bond between the cells. In normal tissues, desmosomes help to maintain the structural integrity of the tissue by holding the cells together. During carcinogenesis, the structure and function of desmosomes may be altered. For example, in oral cancer, the expression of certain cadherins may be increased, leading to increased cell-cell adhesion and a more cohesive tumour mass. This may contribute to the ability of cancer cells to evade the immune system and resist chemotherapy. In addition to their role in cell adhesion, desmosomes also play a role in cell signaling. The proteins that make up desmosomes can interact with signaling pathways that regulate cell proliferation, migration and survival. Dysregulation of these pathways may contribute to the development and progression of oral cancer. There is also evidence that desmosomes may be involved in the process of invasion and metastasis, which is the spread of cancer cells from the primary tumour to other parts of the body. Cancer cells that have disrupted or abnormal desmosomes may be more likely to migrate and invade other tissues. Overall, desmosomes appear to be important in the development and progression of oral cancer. Further research is needed to fully understand the role of these cell-cell junctions in the disease and to identify potential therapeutic targets.

Chronic diastolic stretch unmasks conduction defects in an in vitro model of arrhythmogenic cardiomyopathy.

We seek to elucidate the precise nature of mechanical loading that precipitates conduction deficits in a concealed-phase model of arrhythmogenic cardiomyopathy (ACM). ACM is a progressive disorder often resulting from mutations in desmosomal proteins. Exercise has been shown to worsen disease progression and unmask arrhythmia vulnerability, yet the underlying pathomechanisms may depend on the type and intensity of exercise. Because exercise causes myriad changes to multiple inter-dependent hemodynamic parameters, it is difficult to isolate its effects to specific changes in mechanical load. Here, we use engineered heart tissues (EHTs) with iPSC-derived cardiomyocytes expressing R451G desmoplakin, an ACM-linked mutation, which results in a functionally null model of desmoplakin (DSP). We also use a novel bioreactor to independently perturb tissue strain at different time points during the cardiac cycle. We culture EHTs under three strain regimes: normal physiological shortening; increased diastolic stretch, simulating high preload; and isometric culture, simulating high afterload. DSPR451G EHTs that have been cultured isometrically undergo adaptation, with no change in action potential parameters, conduction velocity, or contractile function, a phenotype confirmed by global proteomic analysis. However, when DSPR451G EHTs are subjected to increased diastolic stretch, they exhibit concomitant reductions in conduction velocity and the expression of connexin-43. These effects are rescued by inhibition of both lysosome activity and ERK signaling. Our results indicate that the response of DSPR451G EHTs to mechanical stimuli depends on the strain and the timing of the applied stimulus, with increased diastolic stretch unmasking conduction deficits in a concealed-phase model of ACM.

Arrhythmogenic Cardiomyopathy: from Preclinical Models to Genotype-phenotype Correlation and Pathophysiology.

Arrhythmogenic cardiomyopathy (ACM) is a hereditary myocardial disease characterized by the replacement of the ventricular myocardium with fibrous fatty deposits. ACM is usually inherited in an autosomal dominant pattern with variable penetrance and expressivity, which is mainly related to ventricular tachyarrhythmia and sudden cardiac death (SCD). Importantly, significant progress has been made in determining the genetic background of ACM due to the development of new techniques for genetic analysis. The exact molecular pathomechanism of ACM, however, is not completely clear and the genotype-phenotype correlations have not been fully elucidated, which are useful to predict the prognosis and treatment of ACM patients. Different gene-targeted and transgenic animal models, human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) models, and heterologous expression systems have been developed. Here, this review aims to summarize preclinical ACM models and platforms promoting our understanding of the pathogenesis of ACM and assess their value in elucidating the ACM genotype-phenotype relationship.

Plakophilin-3 Binds the Membrane and Filamentous Actin without Bundling F-Actin.

Plakophilin-3 is a ubiquitously expressed protein found widely in epithelial cells and is a critical component of desmosomes. The plakophilin-3 carboxy-terminal domain harbors nine armadillo repeat motifs with largely unknown functions. Here, we report the 5 Å cryogenic electron microscopy (cryoEM) structure of the armadillo repeat motif domain of plakophilin-3, one of the smaller cryoEM structures reported to date. We find that this domain is a monomer or homodimer in solution. In addition, using an in vitro actin co-sedimentation assay, we show that the armadillo repeat domain of plakophilin-3 directly interacts with F-actin. This feature, through direct interactions with actin filaments, could be responsible for the observed association of extra-desmosomal plakophilin-3 with the actin cytoskeleton directly attached to the adherens junctions in A431 epithelial cells. Further, we demonstrate, through lipid binding analyses, that plakophilin-3 can effectively be recruited to the plasma membrane through phosphatidylinositol-4,5-bisphosphate-mediated interactions. Collectively, we report on novel properties of plakophilin-3, which may be conserved throughout the plakophilin protein family and may be behind the roles of these proteins in cell-cell adhesion.

Myocardial Inflammation as a Manifestation of Genetic Cardiomyopathies: From Bedside to the Bench.

Over recent years, preclinical and clinical evidence has implicated myocardial inflammation (M-Infl) in the pathophysiology and phenotypes of traditionally genetic cardiomyopathies. M-Infl resembling myocarditis on imaging and histology occurs frequently as a clinical manifestation of classically genetic cardiac diseases, including dilated and arrhythmogenic cardiomyopathy. The emerging role of M-Infl in disease pathophysiology is leading to the identification of druggable targets for molecular treatment of the inflammatory process and a new paradigm in the field of cardiomyopathies. Cardiomyopathies constitute a leading cause of heart failure and arrhythmic sudden death in the young population. The aim of this review is to present, from bedside to bench, the current state of the art about the genetic basis of M-Infl in nonischemic cardiomyopathies of the dilated and arrhythmogenic spectrum in order to prompt future research towards the identification of novel mechanisms and treatment targets, with the ultimate goal of lowering disease morbidity and mortality.

Cheek-Pro-Heart: What Can the Buccal Mucosa Do for Arrhythmogenic Cardiomyopathy?

Arrhythmogenic cardiomyopathy (ACM) is a heart muscle disease associated with ventricular arrhythmias and a high risk of sudden cardiac death (SCD). Although the disease was described over 40 years ago, its diagnosis is still difficult. Several studies have identified a set of five proteins (plakoglobin, Cx43, Nav1.5, SAP97 and GSK3ß), which are consistently re-distributed in myocardial samples from ACM patients. Not all protein shifts are specific to ACM, but their combination has provided us with a molecular signature for the disease, which has greatly aided post-mortem diagnosis of SCD victims. The use of this signature, however, was heretofore restricted in living patients, as the analysis requires a heart sample. Recent studies have shown that buccal cells behave similarly to the heart in terms of protein re-localization. Protein shifts are associated with disease onset, deterioration and favorable response to anti-arrhythmic therapy. Accordingly, buccal cells can be used as a surrogate for the myocardium to aid diagnosis, risk stratification and even monitor response to pharmaceutical interventions. Buccal cells can also be kept in culture, hence providing an ex vivo model from the patient, which can offer insights into the mechanisms of disease pathogenesis, including drug response. This review summarizes how the cheek can aid the heart in the battle against ACM.

Dsg3 epitope-specific signalling in pemphigus.

Introduction: Pemphigus is an autoantibody driven disease that impairs the barrier function of the skin and mucosa by disrupting desmosomes and thereby impeding cellular cohesion. It is known that the different clinical phenotypes of pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are dependent on the autoantibody profile and target antigens that, amongst others, are primarily desmoglein (Dsg)1 and/or Dsg3 for PV and Dsg1 for PF. However, it was reported that autoantibodiesagainst different epitopes of Dsg1 and Dsg3 can be pathogenic or not. The underlying mechanisms are very complex and involve both direct inhibition of Dsg interactions and downstream signalling. The aim of this study was to find out whether there is target-epitope-specific Dsg3 signalling by comparing the effects of the two pathogenic murine IgGs, 2G4 and AK23. Methods: Dispase-based dissociation assay, Western Blot analysis, Stimulated emission depletion microscopy, Fura-based Ca2+ flux measurements, Rho/Rac G-Protein-linked immunosorbent assay, Enzyme-linked immunosorbent assay. Results: The IgGs are directed against the EC5 and EC1 domain of Dsg3, respectively. The data show that 2G4 was less effective in causing loss of cell adhesion, compared to AK23. STED imaging revealed that both autoantibodies had similar effects on keratin retraction and reduction of desmosome number whereas only AK23 induced Dsg3 depletion. Moreover, both antibodies induced phosphorylation of p38MAPK and Akt whereas Src was phosphorylated upon treatment with AK23 only. Interestingly, Src and Akt activation were p38MAPK-dependent. All pathogenic effects were rescued by p38MAPK inhibition and AK23-mediated effects were also ameliorated by Src inhibition. Discussion: The results give first insights into pemphigus autoantibody-induced Dsg3 epitope-specific signalling which is involved in pathogenic events such as Dsg3 depletion.

Erratum: SIRT1 activation and its effect on intercalated disc proteins as a way to reduce doxorubicin cardiotoxicity.

[This corrects the article DOI: 10.3389/fphar.2022.1035387.].

Alterations in Calcium Handling Are a Common Feature in an Arrhythmogenic Cardiomyopathy Cell Model Triggered by Desmosome Genes Loss.

Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disease characterized by fibrofatty replacement of the myocardium. Deleterious variants in desmosomal genes are the main cause of ACM and lead to common and gene-specific molecular alterations, which are not yet fully understood. This article presents the first systematic in vitro study describing gene and protein expression alterations in desmosomes, electrical conduction-related genes, and genes involved in fibrosis and adipogenesis. Moreover, molecular and functional alterations in calcium handling were also characterized. This study was performed d with HL1 cells with homozygous knockouts of three of the most frequently mutated desmosomal genes in ACM: PKP2, DSG2, and DSC2 (generated by CRISPR/Cas9). Moreover, knockout and N-truncated clones of DSP were also included. Our results showed functional alterations in calcium handling, a slower calcium re-uptake was observed in the absence of PKP2, DSG2, and DSC2, and the DSP knockout clone showed a more rapid re-uptake. We propose that the described functional alterations of the calcium handling genes may be explained by mRNA expression levels of ANK2, CASQ2, ATP2A2, RYR2, and PLN. In conclusion, the loss of desmosomal genes provokes alterations in calcium handling, potentially contributing to the development of arrhythmogenic events in ACM.

Neurodegeneration in a novel invertebrate model system: Failed microtubule-mediated cell adhesion and unraveling of macroglia.

Neurodegenerative diseases are among the main causes of death in the United States, leading to irreversible disintegration of neurons. Despite intense international research efforts, cellular mechanisms that initiate neurodegeneration remain elusive, thus inhibiting the development of effective preventative and early onset medical treatment. To identify underlying cellular mechanisms that initiate neuron degeneration, it is critical to identify histological and cellular hallmarks that can be linked to underlying biochemical processes. Due to the poor tissue preservation of degenerating mammalian brain tissue, our knowledge regarding histopathological hallmarks of early to late degenerative stages is only fragmentary. Here, we introduce a novel model organism to study histological hallmarks of neurodegeneration, the spider Cupiennius salei. We utilized toluidine blue-stained 0.9-µm serial semithin and 50-nm ultrathin sections of young and old spider nervous tissue. Our findings suggest that the initial stages of neurodegeneration in spiders may be triggered by (1) dissociation of neuron- and glia-derived microtubules, and (2) the weakening of microtubule-associated desmosomal junctions that lead to the unraveling of neuron-insulating macroglia, compromising the structural integrity of affected neurons. The involvement of macroglia in the disposal of neuronal debris described here-although different in the proposed transport mechanisms-shows resemblance to the mammalian glymphatic system. We propose that this model system is highly suitable to investigate invertebrate neurodegenerative processes from early onset to scar formation and that this knowledge may be useful for the study of neurodegeneration in mammalian tissue.

Significance of anti-desmocollin autoantibodies in pemphigus.

The major autoantigens for pemphigus are desmogleins (Dsgs), cell-cell adhesive structure proteins, one of the desmosomal cadherins. Recent progress in molecular biology has revealed that IgG autoantibodies of classical pemphigus react with Dsg1 or Dsg3. Desmocollins (Dscs) also belong to the cadherin supergene family that provides structure to the desmosomes and play an important role in cell-to-cell adhesion. In addition to the presence of four desmosomal Dsg isoforms, i.e. Dsg1-4, Dsc1, 2 and 3, all of which are derived from different genes, Dsc1 has been previously identified as the target antigen of IgA autoantibodies in the subcorneal pustular dermatosis (SPD)-type of intercellular IgA dermatosis. In addition to the IgA anti-Dsc1 autoantiboides, the presence of IgG anti-Dsc autoantibodies is described in patients of some autoimmune bullous diseases. In particular, the current pemphigus detecting autoantibodies to Dscs has shown a tendency in atypical variants of pemphigus. Therefore, autoantibodies against Dscs alone may cause detachment of cell-cell adhesion in the epidermis in some pemphigus. However, except for the findings of a few in vitro and in vivo studies, there is currently no clear evidence for the pathogenicity of anti-Dsc autoantibodies in pemphigus, whereas significance of anti-Dsg autoantibodies is well established. This article describes the structure and function of the Dscs, and explores the evidence regarding the pathogenic role of anti-Dsc autoantibodies in pemphigus.

Plakophilin 2 regulates intestinal barrier function by modulating protein kinase C activity in vitro.

Previous data provided evidence for a critical role of desmosomes to stabilize intestinal epithelial barrier (IEB) function. These studies suggest that desmosomes not only contribute to intercellular adhesion but also play a role as signaling hubs. The contribution of desmosomal plaque proteins plakophilins (PKP) in the intestinal epithelium remains unexplored. The intestinal expression of PKP2 and PKP3 was verified in human gut specimens, human intestinal organoids as well as in Caco2 cells whereas PKP1 was not detected. Knock-down of PKP2 using siRNA in Caco2 cells resulted in loss of intercellular adhesion and attenuated epithelial barrier. This was paralleled by changes of the whole desmosomal complex, including loss of desmoglein2, desmocollin2, plakoglobin and desmoplakin. In addition, tight junction proteins claudin1 and claudin4 were reduced following the loss of PKP2. Interestingly, siRNA-induced loss of PKP3 did not change intercellular adhesion and barrier function in Caco2 cells, while siRNA-induced loss of both PKP2 and PKP3 augmented the changes observed for reduced PKP2 alone. Moreover, loss of PKP2 and PKP2/3, but not PKP3, resulted in reduced activity levels of protein kinase C (PKC). Restoration of PKC activity using Phorbol 12-myristate 13-acetate (PMA) rescued loss of intestinal barrier function and attenuated the reduced expression patterns of claudin1 and claudin4. Immunostaining, proximity ligation assays and co-immunoprecipitation revealed a direct interaction between PKP2 and PKC. In summary, our in vitro data suggest that PKP2 plays a critical role for intestinal barrier function by providing a signaling hub for PKC-mediated expression of tight junction proteins claudin1 and claudin4.

Structure and regulation of desmosomes in intercalated discs: Lessons from epithelia.

For electromechanical coupling of cardiomyocytes, intercalated discs (ICDs) are pivotal as highly specialized intercellular contact areas. ICD consists of adhesive contacts, such as desmosomes and adherens junctions (AJs) that are partially intermingled and thereby form an area composita to provide mechanical strength, as well as gap junctions (GJ) and sodium channels for excitation propagation. In contrast, in epithelia, mixed junctions with features of desmosomes and AJs are regarded as transitory primarily during the formation of desmosomes. The anatomy of desmosomes is defined by a typical ultrastructure with dense intracellular plaques anchoring the cadherin-type adhesion molecules to the intermediate filament cytoskeleton. Desmosomal diseases characterized by impaired adhesive and signalling functions of desmosomal contacts lead to arrhythmogenic cardiomyopathy when affecting cardiomyocytes and cause pemphigus when manifesting in keratinocytes or present as cardiocutaneous syndromes when both cell types are targeted by the disease, which underscores the high biomedical relevance of these cell contacts. Therefore, comparative analyses regarding the structure and regulation of desmosomal contacts in cardiomyocytes and epithelial cells are helpful to better understand disease pathogenesis. In this brief review, we describe the structural properties of ICD compared to epithelial desmosomes and suggest that mechanisms regulating adhesion may at least in part be comparable. Also, we discuss whether phenomena such as hyperadhesion or the bidirectional regulation of desmosomes to serve as signalling hubs in epithelial cells may also be relevant for ICD.

Key Factors in the Complex and Coordinated Network of Skin Keratinization: Their Significance and Involvement in Common Skin Conditions.

The epidermis serves many vital roles, including protecting the body from external influences and healing eventual injuries. It is maintained by an incredibly complex and perfectly coordinated keratinization process. In this process, desquamation is essential for the differentiation of epidermal basal progenitor cells into enucleated corneocytes, which subsequently desquamate through programmed death. Numerous factors control keratinocyte differentiation: epidermal growth factor, transforming growth factor-α, keratinocyte growth factor, interleukins IL-1-ß and IL-6, elevated vitamin A levels, and changes in Ca2+ concentration. The backbone of the keratinocyte transformation process from mitotically active basal cells into fully differentiated, enucleated corneocytes is the expression of specific proteins and the creation of a Ca2+ and pH gradient at precise locations within the epidermis. Skin keratinization disorders (histologically characterized predominantly by dyskeratosis, parakeratosis, and hyperkeratosis) may be categorized into three groups: defects in the α-helical rod pattern, defects outside the α-helical rod domain, and disorders of keratin-associated proteins. Understanding the process of keratinization is essential for the pathogenesis of many dermatological diseases because improper desquamation and epidermopoiesis/keratinization (due to genetic mutations of factors or due to immune pathological processes) can lead to various conditions (ichthyoses, palmoplantar keratodermas, psoriasis, pityriasis rubra pilaris, epidermolytic hyperkeratosis, and others).

The N-Terminal Part of the 1A Domain of Desmin Is a Hot Spot Region for Putative Pathogenic DES Mutations Affecting Filament Assembly.

Desmin is the major intermediate filament protein of all three muscle cell types, and connects different cell organelles and multi-protein complexes such as the cardiac desmosomes. Several pathogenic mutations in the DES gene cause different skeletal and cardiac myopathies. However, the significance of the majority of DES missense variants is currently unknown, since functional data are lacking. To determine whether desmin missense mutations within the highly conserved 1A coil domain cause a filament assembly defect, we generated a set of variants with unknown significance and systematically analyzed the filament assembly using confocal microscopy in transfected SW-13, H9c2 cells and cardiomyocytes derived from induced pluripotent stem cells. We found that mutations in the N-terminal part of the 1A coil domain affect filament assembly, leading to cytoplasmic desmin aggregation. In contrast, mutant desmin in the C-terminal part of the 1A coil domain forms filamentous structures comparable to wild-type desmin. Our findings suggest that the N-terminal part of the 1A coil domain is a hot spot for pathogenic desmin mutations, which affect desmin filament assembly. This study may have relevance for the genetic counselling of patients carrying variants in the 1A coil domain of the DES gene.